THALLUS MORPHOLOGY AND SPORE FORMATION IN CULTURED ERYTHROCLADIA IRREGULARIS ROSENVINGE (RHODOPHYTA,BANGIOPHYCIDAE)1

The discoid thallus of Erythrocladia irregularis is the result of a pattern of cellular divisions expressed by mono-and endospores; the latter are described for the first time in Erythrocladia. In nutrient enriched growth conditions the discoid thallus becomes a shapeless aggregate of unicells with a wrinkled wall surface, whose plane of division is unpredictable. The cells are able to produce monosporangia with a curved wall or they form eight-celled endosporangia. Chloroplasts have an internal pyrenoid, as is characteristic of the Erythropeltidaceae. The observation that E. irregularis can form either a colonial cluster of unicells or a discoid thallus would lead to the assignment of the genus to the Porphyridiales; however, since this order is reserved to organisms in which only the unicellular organization can be expressed, it is suggested to maintain Erythrocladia in the Erythropeltidales.     

Production of membrane proteins for NMR studies using the condensed single protein (cSPP) production system

In the Single Protein Production (SPP) method, all E. coli cellular mRNAs are eliminated by the induction of MazF, an ACA-specific mRNA interferase. When an mRNA for a membrane protein, engineered to have no ACA sequences without altering its amino acid sequence, is induced in the MazF-induced cells, E. coli is converted into a bioreactor producing only the targeted membrane protein. Here we demonstrate that three prokaryotic inner membrane proteins, two prokaryotic outer membrane proteins, and one human virus membrane protein can be produced at very high levels, and assembled in appropriate membrane fractions. The condensed SPP (cSPP) system was used to selectively produce isotope-enriched membrane proteins for NMR studies in up to 150-fold condensed culture without affecting protein yields, providing more than 99% cost saving for isotopes. As a novel application of the cSPP system for studies of membrane proteins prior to purification we also demonstrate, for the first time, fast detergent screening by microcoil NMR and well-resolved NMR spectra of several targeted integral membrane proteins obtained without purification.     

Phylogenetic conservation of cytostatic factor related genes in the ascidian Ciona intestinalis

In all vertebrates, mature oocytes arrest at the metaphase of the II meiotic division, while some invertebrates arrest at metaphase-I, others at prophase-I. Fertilization induces completion of meiosis and entry into the first mitotic division. Several experimental models have been considered from both vertebrates and invertebrates in order to shed light on the peculiar aspects of meiotic division, such as the regulation of the cytostatic factor (CSF) and the maturation promoting factor (MPF) in metaphase I or II. Recently, we proposed the oocytes of ascidian Ciona intestinalis as a new model to study the meiotic division. Here, taking advantage of the recent publication of the C. intestinalis genome, we presented a phylogenetic analysis of key molecular components of the CSF-related machinery. We showed that the Mos/MAP kinase pathway is perfectly conserved in ascidians. We demonstrated the presence of a CSF-like activity in metaphase-I arrested C. intestinalis oocytes able to block cell division in two-cell embryos. We further investigated the regulation of CSF by demonstrating that both CSF and MPF inactivation, at the exit of metaphase-I, are independent from protein synthesis, indicating the absence of short-lived factors that regulate metaphase stability, as in other invertebrate species. The results obtained suggest that meiotic regulation in C. intestinalis resembles that of vertebrates, such as Xenopus accordingly to the position of this organism in the evolutionary tree.     

Coronary artery bypass with glycerol-preserved saphenous vein allografts

Over a 2-year period, 19 patients whose autologous saphenous veins were either unsuitable or unavailable underwent myocardial revascularization with saphenous vein allografts (SVAs) at our institution. All SVAs had been preserved in 98% glycerol at room temperature for at least 3 weeks (average, 7 weeks); before use, they were rinsed with saline and antibiotic solution. One operative death (5.2%) and three late deaths (16.6%) occurred. Fourteen of the long-term survivors have been observed for 24 to 48 months (average 32.2 months) postoperatively. Nine are asymptomatic, whereas four complain of angina, and one reports exertional dyspnea with-out chest pain. Only three patients have been restudied (7, 10, and 18 months after surgery, respectively); in each of these patients, angiography has shown occlusion of all SVAs. However, histologic examination of glycerol-preserved SVAs has not revealed pathologic changes that would suggest potential graft failure. Despite fairly satisfactory clinical results, preliminary hemodynamic data indicate that glycerol-preserved SVAs are unsuitable for myocardial revascularization in the absence of autologous saphenous veins.     

Computational Design of a Protein-Based Enzyme Inhibitor

While there has been considerable progress in designing protein–protein interactions, the design of proteins that bind polar surfaces is an unmet challenge. We describe the computational design of a protein that binds the acidic active site of hen egg lysozyme and inhibits the enzyme. The design process starts with two polar amino acids that fit deep into the enzyme active site, identifies a protein scaffold that supports these residues and is complementary in shape to the lysozyme active-site region, and finally optimizes the surrounding contact surface for high-affinity binding. Following affinity maturation, a protein designed using this method bound lysozyme with low nanomolar affinity, and a combination of NMR studies, crystallography, and knockout mutagenesis confirmed the designed binding surface and orientation. Saturation mutagenesis with selection and deep sequencing demonstrated that specific designed interactions extending well beyond the centrally grafted polar residues are critical for high-affinity binding.     

Density gradient centrifugation of sperm from a subfertile stallion and effect of seminal plasma addition on fertility

Stallions are not selected for fertility but for other criteria (pedigree, conformation, performances, progeny), therefore valuable but subfertile stallions with poor semen quality are frequently used in commercial breeding programs. The object of this study was to evaluate whether sperm selection through a silane-coated silica colloid gradient centrifugation, with or without the addition of seminal plasma of a high fertile stallion, could improve the pregnancy rates of an oligospermic valuable stallion in a commercial breeding program. In 2008 breeding season (experiment 1, n=104 mares), simple centrifugation and density gradient centrifugation of the sperm were compared. In 2009 and 2010 breeding seasons (experiment 2, n=125 mares), the effect of the addition of 5% seminal plasma to the extender after sperm selection was evaluated. In all mares deep horn uterine insemination was performed with 1 ml containing 50×10(6) morphologically normal progressive motile spermatozoa, 24-30 h after induction of ovulation with hCG. Pregnancy diagnosis by ultrasonography was performed 14 days following ovulation. Results showed a higher per cycle pregnancy rate (P>0.05) when sperm selection through a density gradient was used (62% vs. 42.3%, exp 1), while the addition of 5% seminal plasma did not influence the outcome (45.9% vs. 47.6%, exp 2) (P>0.05). An age-related decrease in the fertility of the stallion was observed when comparing the results from the different breeding seasons (P<0.05). In conclusion, sperm selection through a discontinuous density gradient enabled a normal per cycle pregnancy rate to be achieved from an oligospermic-subfertile stallion in a commercial breeding program, and no differences were observed regarding the addition of seminal plasma.